Characterizing clearance mechanisms of TDP-43 and disease associated isoforms

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. Over 95% of ALS patients exhibit cytoplasmic mislocalization and aggregation of the nuclear RNA-binding protein, TDP-43. These aggregates often contain N-terminal or C- terminal truncated isoforms of TDP-43 along with full length TDP-43 (flTDP-43). It is unclear if these aggregates are the direct cause of cell death, though reducing cytoplasmic TDP-43 or its isoforms in disease models improves cell viability. Recently, our lab uncovered a macroautophagy-independent endolysosomal mechanism that requires the E3 ubiquitin ligase, Rsp5/NEDD4, the ubiquitin segregase chaperone, Cdc48/VCP, and ESCRT machinery to facilitate trafficking of TDP-43 into multivesicular bodies (MVBs). However, many mechanistic steps in this process remain unclear including how this pathway relates to a previously defined means of endolysosomal cytoplasmic protein degradation known as endosomal microautophagy (eMI). Using biochemical, genetic, and microscopy-based approaches, I will determine the specific interactions, modifications, and cellular conditions under which TDP-43 is targeted to MVBs in human and neuronal-cell models. These experiments will uncover new mechanistic detail regarding TDP-43 endolysosomal degradation and eMI. I will also investigate the degradation mechanism of three TDP-43 isoforms: sTDP-43, TDP-35, and TDP-25. TDP-43 isoforms have distinct half-lives from flTDP-43 which suggests isoform-specific degradation pathways may exist. Targeted degradation of disease-specific TDP-43 isoforms may have greater efficacy in disease treatment, while preserving functional flTDP-43 species thus avoiding loss of function phenotypes. Using our unbiased genome- wide dot blot screening platform in yeast and candidate-based approaches based on our current work, we will elucidate the clearance mechanisms of all major TDP-43 isoforms. Collectively, this study will uncover new mechanistic detail regarding TDP-43 endolysosomal degradation, reveal broader insight into cytoplasmic proteostasis mechanisms, and advance the search for novel therapeutic targets for ALS and other TDP-43 proteinopathies.